Altered Apoptotic Responses in Chemically-Resistant Mammalian Cell Variants

在所有多细胞生物体中，组织和器官的稳态是通过细胞增殖和细胞死亡之间的严格平衡来维持的。 细胞死亡的最重要机制之一是被称为细胞凋亡的基因调控的细胞自杀过程。 该过程的几种形式已显示涉及（a）凋亡抑制因子Bcl-2，（B）因子Bid，其在活化时诱导细胞色素c从线粒体流出，和（c）几种特异性蛋白酶，包括半胱天冬酶-3和半胱天冬酶-8。 本项目的总体目的是阐明这些基因产物在星形孢菌素诱导人HL-60培养细胞凋亡模型系统中的分子和生化作用。 实验将回答三个具体问题。(1)处理Bid的星形孢菌素激活的caspase是什么？ 半胱天冬酶将被生物化学纯化。 如果是新的，它将被测序，其基因将被克隆。 (2)Bcl-2如何调节星形孢菌素诱导的caspase-8激活？ 将考虑几种可能性，包括与其他组分形成信号复合物。(2)Bcl-2在胞质溶胶中与Bid形成异二聚体并影响Bid对caspase作用的敏感性吗？ 将直接测定此类二聚体的可能形成及其底物活性。 预计这些结果将产生适用于各种多细胞生物的细胞凋亡的基本新信息。

Tissue and organ homeostasis in all multicellular organisms is maintained by a rigid balance between cellular proliferation and cell death. One of the most important mechanisms of cell death is a genetically regulated process of cellular suicide known as apoptosis. Several forms of this process have been shown to involve (a) the apoptotic suppressor, Bcl-2, (b) a factor, Bid, which when activated induces cytochrome c efflux from mitochondria, and (c) several specific proteases, including caspase-3 and caspase-8. The overall purpose of this project is to elucidate the molecular and biochemical roles of these gene products in the model system of apoptosis induced by staurosporine in human HL-60 cultured cells. Experiments will be undertaken to answer three specific questions. (1) What is the staurosporine-activated caspase that processes Bid? The caspase will be purified biochemically. If novel, it will be sequenced, and its gene will be cloned. (2) How does Bcl-2 regulate staurosporine-induced caspase-8 activation? Several possibilities will be considered including the formation of signaling complexes with other components. (2) Does Bcl-2 form heterodimers with Bid in the cytosol and affect the susceptibility of Bid to caspase action? The possible formation of such dimers and their substrate activities will be assayed directly. It is expected that the results will yield fundamental new information about apoptosis applicable to a wide variety of multicellular organisms.